Genetically Modified Non-Human Animals And Methods Of Use Thereof

ABSTRACT

The invention relates generally to genetically modified non-human animals expressing human polypeptides and their methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/698,002, filed Sep. 7, 2012, and to U.S. Provisional ApplicationSer. No. 61/775,171, filed Mar. 8, 2013, the contents of each of whichare incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

The aim of biomedical research is to gain a better understanding ofhuman physiology and to use this knowledge to prevent, treat or curehuman diseases. Due to practical and ethical barriers to theexperimentation on human subjects, many studies are conducted on smallanimal models, such as the mouse. However, mice are not people and theknowledge gained from animal experimentation is not always applicable tohumans. In this context, mice repopulated with a human hemato-lymphoidsystem (HHLS) represent a useful small animal model for the study ofhuman hematopoiesis and immune function in vivo.

HHLS mice are generated by the transplantation of human hematopoieticstem and progenitor cells (HSPCs) and/or human fetal tissues intorecipient mice deficient in the innate and adaptive arms of the immuneresponse. The first models of HHLS mice were developed in the late 1980s(Mosier et al., 1988, Nature 335:256-259; McCune et al., 1988, Science241:1632-1639; Kamel-Reid and Dick, 1988, Science 242:1706-1709), andhave been undergoing a series of improvements since then (Legrand etal., 2006, Journal of Immunology 176:2053-2058; Shultz et al., 2007,Nature Reviews Immunology 7:118-130). The strains of mice currently usedas recipients for human hematopoietic engraftment share threecharacteristics. First, they lack B and T cells due to the Scid mutationin the gene encoding the PRKDC protein (Mosier et al., 1988, Nature335:256-259; McCune et al., 1988, Science 241:1632-1639), or due todeletion of one of the two Rag genes (Shultz et al., 2000, Journal ofimmunology 164:2496-2507; Traggiai et al., 2004, Science 304:104-107).Second, deletion or mutation of the Il2rg gene that encodes the commongamma chain (γ_(c)) of cytokine receptors abolishes IL-15 signaling andresults in the absence of NK cells (Traggiai et al., 2004, Science304:104-107; Ito et al. 2002, Blood 100:3175-3182). Third, theinteraction between the SIRPA receptor expressed on mouse macrophagesand the CD47 ligand on human cells provides an inhibitory signal tomouse macrophages and confers phagocytic tolerance for the humanxenograft (Takenaka et al., 2007, Nature Immunology 8:1313-1323;Takizawa & Manz, 2007, Nature Immunology 8:1287-1289). Cross-speciesinteraction between SIRPA expressed on mouse cells and human CD47 isachieved when using the NOD genetic background which contains a naturalpolymorphism in the Sirpa gene (Takenaka et al., 2007, Nature Immunology8:1313-1323; Takizawa & Manz, 2007, Nature Immunology 8:1287-1289;Legrand et al., 2011, Proc Natl Acad Sci USA 108:13224-13229) or byBAC-transgenic expression of the human SIRPA gene (Strowig et al., 2011,Proc Natl Acad Sci USA 108:13218-13223). High levels of humanhematopoietic cell engraftment, upon human HSPC transplantation, areachieved when using NOD Scid γ_(c) ^(−/−) (NOG (Ito et al. 2002, Blood100:3175-3182) or NSG (Ishikawa et al., 2005, Blood 106:1565-1573)) orhSIRPA^(tg) RAG2^(−/−) γ_(c) ^(−/−) (SRG (Strowig et al., 2011, ProcNatl Acad Sci USA 108:13218-13223)) mice as recipients.

Although human multi-lineage hematopoietic development is observed inthese recipient strains, the terminal differentiation, homeostasisand/or effector function of most human cell types is sub-optimal. It hasbeen hypothesized that this condition is due to reduced or absentcross-reactivity between cytokines secreted by mouse tissues and thehuman receptors expressed on hematopoietic cells (Manz, 2007, Immunity26:537-541; Willinger et al., 2011, Trends in Immunology 32:321-327). Tocircumvent this limitation, several strategies have been developed todeliver human cytokines in the mouse host. These methods include theinjection of recombinant cytokines (Lapidot et al., 1992, Science255:1137-1141; van Lent et al., 2009, J. Immunol 183:7645-7655),lentiviral delivery of cytokine-encoding cDNA (O'Connell et al., 2010,PloS One 5(8):e12009), hydrodynamic injection of plasmid DNA (Chen etal., 2009, Proc Natl Acad Sci USA 106:21783-21788), transgenicexpression of cDNA (Nicolini et al., et al., 2004, Leukemia18(2):341-347; Brehm et al., 2012, Blood 119:2778-2788; Takagi et al.,2012, Blood 119:2768-2777) or knock-in replacement of cytokine-encodinggenes (Rongvaux et al., 2011, Proc Natl Acad Sci USA 108:2378-2383;Willinger et al., 2011, Proc Natl Acad Sci USA 108:2390-2395; Rathinamet al., 2011, Blood 118:3119-3128). The later method has the advantageof more physiological expression of the human gene. Furthermore, if thehuman cytokine is not fully cross-reactive on the mouse receptor, it caninduce a defect in mouse cell populations and confer an additionalcompetitive advantage to human cells. Using a knock-in gene replacementstrategy, humanization of the gene encoding thrombopoietin (Tpo)resulted in better maintenance of functional human hematopoietic stemcells and increased engraftment in the bone marrow (Rongvaux et al.,2011, Proc Natl Acad Sci USA 108:2378-2383); replacement of the genesencoding interleukin-3 and GM-CSF (Il3 and Csf2) induced the loss ofmouse lung alveolar macrophages (AM) and the development of functionalhuman AM (Willinger et al., 2011, Proc Natl Acad Sci USA 108:2390-2395);and substitution of the Csf1 gene, which encodes M-CSF, resulted inincreased numbers of human monocytes in multiple tissues (Rathinam etal., 2011, Blood 118:3119-3128).

Human and mouse hemato-lymphoid systems differ in many aspects (Haley,2003, Toxicology 188:49-71; Mestas & Hughes, 2004, J Immunol172:2731-2738). One of the major differences between the two specieslies in their white blood cell (WBC) differential. Human blood is richin myeloid cells that represent 50-75% of total WBCs. In contrast, mouseblood is dominated by lymphocytes and only 20-30% of WBCs are of myeloidlineages. This species difference, whose functional and evolutionarysignificance is not understood, is not recapitulated in conventionalHHLS mice such as NOG/NSG or SRG. Indeed, human myeloid development isparticularly defective in these hosts, with myeloid cells representingonly 5-10% of human WBCs.

One application of mice with functional human immune systems is thedevelopment and testing of human vaccines. Historically, the inductionof immune responses in vivo has been relatively inefficient (2004,Traggiai et al., Science 304:104-107; 2002, Ito et al., Blood100:3175-3182; 2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultzet al., J Immunol 174:6477-6489; 2006, Baenziger et al., Proc Natl AcadSci USA 103:15951-15956). Several studies have reported successfulpathogen-specific immune responses upon infection. Although it wasreported that around 50% of mice produced virus-specific IgM and IgGupon dengue virus infection (2007, Kuruvilla et al. Virology369:143-152), other studies reported frequencies below 20% of miceproducing antigen-specific IgM and IgG after HIV and EBV infection(2006, Baenziger et al., Proc Natl Acad Sci USA 103:15951-15956; 2008,Yajima et al., J Infect Dis 198:673-682). Upon immunization withadjuvant and antigen, class switching of antigen-specificimmunoglobulins is also historically inefficient with only a fraction ofimmunized animals showing antigen specific IgG responses (2004, Traggiaiet al., Science 304:104-107; 2002, Ito et al., Blood 100:3175-3182;2005, Ishikawa et al., Blood 106:1565-1573; 2005, Shultz et al., JImmunol 174:6477-6489; 2009, Watanabe et al., Int Immunol 21:843-858;2010, Becker et al., PLoS ONE 5). These studies included NSG and BALB/cRAG2^(−/−) γ_(c) ^(−/−) mice and different adjuvant/antigencombinations.

There is a need in the art for humanized non-human animals able tosupport and sustain engraftment with human hematopoietic cells. Thepresent invention addresses this unmet need in the art.

SUMMARY OF THE INVENTION

The invention relates generally to genetically modified non-humananimals expressing at least one of human M-CSF, human IL-3, humanGM-CSF, human SIRPA or human TPO, as well as to their methods of use.Thus, in one embodiment, the invention is a genetically modifiednon-human animal comprising a genome comprising at least one nucleicacid encoding at least one of the group consisting of human M-CSF, humanIL-3, human GM-CSF, human SIRPA and human TPO, where the at least onenucleic acid is operably linked to a promoter, and where the animalexpresses at least one polypeptide selected from the group consisting ofhuman M-CSF, human IL-3, human GM-CSF, human SIRPA and human TPO. Inanother embodiment, the invention is a genetically modified non-humananimal, comprising a genome comprising a nucleic acid encoding humanM-CSF, a nucleic acid encoding human IL-3, a nucleic acid encoding humanGM-CSF, a nucleic acid encoding human SIRPA and a nucleic acid encodinghuman TPO, where each of the nucleic acids encoding human M-CSF, humanIL-3, human GM-CSF, human SIRPA and human TPO is operably linked to apromoter, and where the animal expresses human M-CSF polypeptide, humanIL-3 polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide andhuman TPO polypeptide. In some embodiments, the genetically modifiednon-human animal is immunodeficient. In some embodiments, thegenetically modified non-human animal does not express recombinationactivating gene 2 (Rag-2−/−). In some embodiments, the geneticallymodified non-human animal does not express IL2 receptor gamma chain(gamma chain−/−). In some embodiments, the genetically modifiednon-human animal does not express Rag-2 and the genetically modifiednon-human animal does not express IL2 receptor gamma chain (Rag-2−/−gamma chain−/−). In some embodiments, the genetically modified non-humananimal is a rodent. In some embodiments, the genetically modifiednon-human animal is a mouse. In one embodiment, the genetically modifiednon-human animal also includes at least one human hematopoietic cell. Inone embodiment, the genetically modified non-human animal also includesat least one human cancer cell. In some embodiments, the human cancercell is a leukemia cell or a melanoma cell.

In another embodiment, the invention is a method of hematopoietic stemand progenitor cell (HSPC) engraftment in a genetically modifiednon-human animal, where the animal expresses at least one of the groupconsisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA andhuman TPO, the method comprising the step of: administering at least oneHSPC to the genetically modified animal expressing at least one of thegroup consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPAand human TPO. In some embodiments, the HSPC is a human HSPC. In oneembodiment, the genetically modified non-human animal is a rodent. Inone embodiment, the genetically modified non-human animal is a mouse. Inone embodiment, the genetically modified non-human animal isimmunodeficient. In one embodiment, the genetically modifiedimmunodeficient non-human animal does not express recombinationactivating gene 2 (Rag-2−/−). In one embodiment, the geneticallymodified immunodeficient non-human animal does not express endogenousIL2 receptor (gamma chain−/−). In one embodiment, the geneticallymodified immunodeficient non-human animal does not express endogenousRag-2 and does not express endogenous gamma chain (Rag-2−/− gammachain−/−). In one embodiment, the genetically modified animal comprisesa human cancer cell. In one embodiment, the human cancer cell is aleukemia cell or a melanoma cell.

In another embodiment, the invention is a genetically modified Rag-2−/−,gamma chain−/− mouse having a genome comprising at least one nucleicacid encoding at least one of the group consisting of human M-CSF, humanIL-3, human GM-CSF, human SIRPA and human TPO, where the at least onenucleic acid is operably linked to at least one promoter, where themouse expresses at least one polypeptide selected from the groupconsisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA andhuman TPO. In one embodiment, the genetically modified non-human animalcomprises a genome having a nucleic acid encoding human M-CSF, a nucleicacid encoding human IL-3, a nucleic acid encoding human GM-CSF, anucleic acid encoding human SIRPA and a nucleic acid encoding human TPO,where each of the nucleic acids encoding human M-CSF, human IL-3, humanGM-CSF, human SIRPA and human TPO is operably linked to a promoter, andwhere the animal expresses human M-CSF polypeptide, human IL-3polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide and humanTPO polypeptide. In one embodiment, the genetically modified non-humananimal is a rodent. In one embodiment, the genetically modifiednon-human animal is a mouse. In one embodiment, the genetically modifiednon-human animal comprises a human hematopoietic cell. In oneembodiment, the genetically modified non-human animal comprises a humancancer cell. In some embodiments, the human cancer cell is a leukemiacell or a melanoma cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1E depict the results of experiments showing that MISTRG micesupport high levels of human hematopoietic engraftment. X-raypre-conditioned newborn mice of the indicated strains were engrafted byintra-hepatic injection of 100,000 human fetal liver-(FL-)CD34⁺ cells.Human engraftment levels (hCD45⁺ cells) were measured in the blood 7-9weeks later, and in the BM 10-12 weeks later. (FIG. 1A) Representativeflow cytometry analysis of the frequency of mouse and human CD45⁺ cellsin the blood and BM of the indicated recipient mice. Numbers next togated areas indicate percentages among total CD45⁺ cells. (FIG. 1B)Combined data of blood engraftment levels (% hCD45⁺ cells) from 19independent experiments. In each experiment, a single FL-CD34⁺ cellsample was split and injected into mice of the respective strains. Eachsymbol represents an individual mouse and the red bars indicate meanvalues (n=56-155; ns, not significant; * p<0.05 Tukey test (see FIGS.6A-6C for a complete statistical analysis). The gray horizontal lineindicates 10% hCD45⁺ cells. (FIG. 1C) Engraftment levels in the BM of arepresentative subset of mice (FIG. 6C) from panel (FIG. 6B) (n=12-16; *p<0.05 Tukey test; see also FIGS. 6D-6E). (FIG. 1D) Representative flowcytometry analysis of hCD45⁺ cell engraftment in the blood and BM 3months after intra-hepatic injection of 200,000 FL-CD34⁺ cells intonon-irradiated newborn MISTRG mice. (FIG. 1E) Human CD45⁺ cellengraftment levels in the blood and BM of MISTRG mice transplanted as in(FIG. 1D) (n=16). In this case, the BM of all mice (including mice withblood hCD45⁺<10%) are shown.

FIGS. 2A-2K depict the results of experiments showing that MISTRG micesupport efficient myeloid development and maintenance in lymphoid andnon-lymphoid tissues. (FIG. 2A) Percentages of human myeloid cells(hCD33⁺) among human hematopoietic cells (hCD45⁺) in the blood of theindicated recipient mice, engrafted as newborns by intra-hepaticinjection of FL-CD34⁺ cells after X-ray preconditioning. Each symbolrepresents an individual mouse and the red bars indicate mean values(n=20-113; statistical analysis is shown in FIG. 7A). (FIG. 2B) HumanWBC composition in the same mice (n=20-113 mice/group; n=8 human donors;error bars indicate SEM). (FIG. 2C) Immunohistological staining of humanmyeloid cells (hCD68⁺) in non-lymphoid tissues of the indicatedrecipient mice. The black bar represents 20 μm, and the images shown arerepresentative of at least three mice analyzed per group. (FIG. 2D andFIG. 2E) Representative flow cytometry analysis (FIG. 2D) andfrequencies (FIG. 2E) of human monocyte subsets, identified byexpression of CD14 and CD16 among hCD45⁺CD33⁺ cells in the blood ofrecipient mice (n=8-12 mice/group; error bars indicate SEM). (FIG. 2Fand FIG. 2G) Cytokine production by human monocytes isolated from the BMof MITRG recipients and stimulated in vitro with LPS (FIG. 2F) or R848(FIG. 2G) (error bars indicate SD of triplicates; representative of 3independent experiments). (FIG. 2H) In vitro phagocytosis ofGFP-expressing E. coli by human cells present in the blood of MITRG mice(n=7). (FIGS. 2I, 2J, 2K) In vivo cytokine production measured by ELISAin the serum or by RT-PCR in the lung of mice treated with LPS (FIG. 2I;90 min, n=15-18), or infected with Listeria monocytogenes (FIG. 2J; day2, n=6-15) or influenza A/PR8 H1N1 (FIG. 2K; day 3, n=3-5). (FIGS. 2A,2J, 2K) p-values calculated by one-way ANOVA followed by Tukey posthoctest (* p<0.05); (FIG. 2I) p-value calculated by unpaired Student'st-test on log 10-transformed values.

FIGS. 3A-3I depict the results of experiments showing that MISTRG miceefficiently support the development and function of human NK cells.(FIG. 3A) Quantitative RT-PCR analysis of human IL-15 and IL-15Rα mRNAexpression in the liver of engrafted NSG, MITRG, and MISTRG mice (n=7-8;p-values calculated by one-way ANOVA; *, p<0.05 Tukey post hoc test).Expression was normalized to mouse Hprt. (FIG. 3B) Quantitative RT-PCRanalysis of human IL-15 and IL-15Rα mRNA expression in human cellpopulations purified from bone marrow of engrafted MITRG (n=4-5, errorbars indicate SEM). Expression was normalized to human HPRT and is shownrelative to hCD14⁺hCD16⁻ cells. (FIG. 3C and FIG. 3D) Representativeflow cytometry analysis (gated on hCD45⁺mCD45⁻ cells, lymphocyte gate;numbers next to outlined areas indicate percentages of cells) (FIG. 3C)and absolute number or frequency (FIG. 3D) of human NK cells(hNKp46⁺hCD3⁻) in engrafted NSG, MITRG, and MISTRG (n=8-16; p-valuescalculated by one-way ANOVA; *, p<0.05 Tukey post hoc test). (FIG. 3E)Absolute number of human liver NK (hNKp46⁺hCD3⁻) and T cells (hCD3⁺,shown as control) from engrafted MISTRG mice either left untreated ortreated for 3 consecutive days with liposome-encapsulated clodronate todeplete phagocytic cells (n=8; p-value calculated by unpaired Student'st-test; ns, not significant). (FIG. 3F) Labeled LCL721.221 (HLA class Inegative) and LCL721.45 (class I positive) cells were injected i.v. in a1:1 ratio, and the proportions of HLA class I positive or negative,among labeled cells recovered 12 hours later in the spleen, were used tocalculate specific NK cell cytotoxicity (n=8, p-value calculated byunpaired student's t-test). (FIG. 3G) Quantitative RT-PCR analysis ofhuman IFNγ mRNA expression in the liver of NSG and MISTRG mice 2 daysafter Listeria infection (n=8-9, p-value calculated by unpairedstudent's t-test). Expression was normalized to mouse Hprt. (FIG. 3H andFIG. 3I) Representative flow cytometry analysis (FIG. 3H) and frequency(FIG. 3I) of IFNγ-expressing and degranulating (CD107a⁺) human liver NKcells from either uninfected or Listeria-infected NSG and MISTRG mice(n=4-11; p-value calculated by one-way ANOVA). Results are combined fromtwo (FIGS. 3A, 3E-3I), three (FIG. 3B), or four (FIGS. 3C, 3D)experiments.

FIGS. 4A-4F depict the results of experiments showing that human myeloidcells in MISTRG infiltrate a tumor and support its growth. The humanmelanoma cell line Me290 was implanted in the flank of engrafted ornon-engrafted NSG and MISTRG mice. Some mice were treated with theVEGF-inhibitor Avastin™ The tumors were measured and dissected foranalysis 11 days later. (FIG. 4A) Infiltration of human hematopoieticcells in the tumor, determined by the expression of mRNA encoding humanhematopoietic (PTPRC, encoding CD45) and myeloid (ITGAM, encoding CD11b)markers (n=6-7; p-value calculated by unpaired Student's t-test). (FIG.4B and FIG. 4D) Representative immunohistochemistry pictures of humanmyeloid cell markers in tumors from NSG, MISTRG and patients. (FIG. 4C)Quantification of the density of CD163⁺ cells (n=3 samples/group, 3slides counted/sample). (FIG. 4E and FIG. 4F) Representative pictures(FIG. 4E) and volume (FIG. 4F) of the tumors in the indicated groups ofmice (n=7-24 mice/group). p-values were calculated by Student's t-test(FIG. 4A) or by one-way ANOVA (FIGS. 4C, 4E) followed by Tukey posthoctest (* p<0.05).

FIG. 5 depicts cytokines involved in HSC function and myeloiddevelopment. Schematic representation of hematopoietic stem celldevelopment into myeloid cells and non-exhaustive list of cytokinesknown to regulate this process. Shading indicates the percentages ofamino acid identity between human and mouse cytokines. The percentage ofamino acid identity is the most objective measure of proteinconservation between species, but it does not always correlate withfunctional inter-species cross-reactivity in vivo. Black rectanglesindicate cytokines that are genetically humanized in MISTRG. HSC,hematopoietic stem cell; MPP, multipotent progenitor; CMP, commonmyeloid progenitor; GMP, granulocyte/macrophage progenitor; MEP,megakaryocyte/erythrocyte progenitor.

FIGS. 6A-6E depict the results of statistical analysis of engraftmentlevels in recipient mice. (FIG. 6A) Statistical analysis (one-way ANOVAfollowed by Tukey post-hoc test; ns, not significant) of the datapresented in FIG. 1A (percentage of hCD45⁺ cells in the blood ofrecipient mice). (FIG. 6B) Numbers of recipient mice that reach anengraftment level of at least 10% hCD45⁺ cells in the blood 7-9 weeksafter transplantation. (FIG. 6C) Blood engraftment levels of the miceused in FIG. 1C for analysis of the BM. (FIG. 6D) Statistical analysis,similar to (FIG. 6A), of the data presented in FIG. 1C (percentage ofhCD45⁺ cells in the BM of recipient mice). (FIG. 6E) Absolute numbers ofhCD45⁺ cells in the BM (2 femurs and 2 tibias) of recipient mice shownin FIG. 1C. The reduced numbers of cells in the BM of MISTRG is due tothe smaller size of the mice at that age (10-12 weekspost-transplantation) and is caused by the first clinical signs ofanemia described in detail in FIGS. 10A-10I.

FIGS. 7A-7H depict the results of experiments assessing enhanced humanmyeloid development in MISTRG mice. (FIG. 7A) Statistical analysis(one-way ANOVA followed by Tukey post-hoc test; ns, not significant) ofthe data presented in FIG. 2A (percentage of hCD33⁺ cells in the bloodof recipient mice). (FIG. 7B and FIG. 7C) Frequencies (FIG. 7B) andstatistical analysis (FIG. 7C) of human myeloid cells (hCD33⁺) in the BMof recipient mice. (FIG. 7D) Representative flow cytometry analysis ofhuman lymphoid and myeloid lineages in the blood of MISTRG. (FIG. 7E andFIG. 7F) Representative flow cytometry analysis of human monocytes(CD33^(hi)SSC^(lo)CD66⁻) and granulocytes (CD33⁺SSC^(hi)CD66⁺) in the BM(FIG. 7E) and blood (FIG. 7F) of MISTRG and human donor. (FIG. 7G andFIG. 7H) Absolute numbers of human myeloid cells (hCD33⁺) in the lung(FIG. 7G) and liver (FIG. 7H) of recipient mice (n=8-12; p-valuescalculated by one-way ANOVA followed by Tukey posthoc test, * p<0.05).

FIGS. 8A and 8B depict the results of experiments showing enhanceddevelopment of human monocyte subsets in MISTRG mice. (FIG. 8A)Representative flow cytometry analysis of human monocyte subsets,identified by expression of CD14 and CD16 among hCD45⁺CD33⁺ cells in theBM, spleen, lung and liver of the indicated recipient mice. (FIG. 8B)Frequencies (error bars represent SEM) among hCD33⁺ cells and absolutenumbers of monocyte subsets in the lung and liver of recipient mice(n=12 mice/group; p-values calculated by one-way ANOVA; *, p<0.05 Tukeypost hoc test).

FIGS. 9A and 9B depict the results of experiments showing that humanmonocyte subsets are similar in MISTRG and in human donors. Extendedimmunophenotype of the indicated subsets of human monocytes in the blood(FIG. 9A) and BM (FIG. 9B) of MISTRG recipients and human donor.Staining with isotype control antibodies and specific antibodies isshown.

FIGS. 10A-10I depict the results of experiments showing that humanmyeloid cells breach human-to-mouse phagocytic tolerance. (FIG. 10A)CFSE-labeled mouse RBCs were transferred into the indicated mice and thefrequency of labeled cells was measured at the indicated time points.(FIG. 10B) Engrafted MISTRG were pre-treated or not with clodronate todeplete phagocytic cells and CFSE-labeled mouse RBCs were transferredand monitored as in (FIG. 10A) (p-value, clodronate-effect measured byrepeated measure ANOVA for days 1-3). These results show thattransferred mouse RBCs are rapidly cleared in vivo by phagocytic cellsthat are present in MISTRG but not in NSG. (FIG. 10C) RBC counts in theblood of non-engrafted mice (n=9-15) or 8-10 weeks after engraftmentwith human FL-CD34⁺ cells (n=11-37). p-values indicate comparisonbetween non-engrafted and engrafted mice of each genotype (Student'sunpaired t test). (FIG. 10D) Correlation between human engraftmentlevels (percentage of hCD45⁺ cells in the blood) and RBC counts(n=13-22). (FIG. 10E) Flow cytometry analysis of mouse (mTer119⁺) andhuman (hCD235a⁺) erythroid cells in the blood of non-engrafted orengrafted MISTRG, showing that almost all erythroid cells in the bloodof engrafted MISTRG are of mouse origin, and human erythroid cells arebarely detectable. (FIG. 10F) Representative pictures and spleen weightof engrafted mice of the indicated strains (n=3-22), showingsplenomegaly in engrafted MISTRG mice. Spleens from Balb/c mice wereused as a control (p-value, one-way ANOVA; *, p<0.05 compared to allother groups, Tukey posthoc test). (FIG. 10G) Histological section ofthe spleen of engrafted NSG and MISTRG stained with H&E, illustratingthe enlargement of the red pulp in MISTRG mice with splenomegaly. (FIG.10H) Flow cytometry analysis of mouse erythroid progenitors(mTer119⁺mCD71⁺), which represent up to 80% of the cells in the spleenof engrafted MISTRG. (FIG. 10I) Blood smears of non-engrafted andengrafted MISTRG illustrate enrichment in reticulocytes. Taken together,these results strongly suggest that anemia in MISTRG results from theabsence of human-to-mouse phagocytic tolerance, and massiveextra-medullary mouse erythropoiesis fails to compensate for thedestruction of mRBCs. Results are representative of at least 5 miceexamined in each group (FIGS. 10C, 10E-10I) and 2 independentexperiments (FIGS. 10A, 10B).

FIGS. 11A and 11B depict the results of experiments showing that MISTRGmice provide human IL-15/IL-15Rα. (FIG. 11A) Quantitative RT-PCRanalysis of human IL-15 and IL-15Rα mRNA expression in the lung ofengrafted NSG, MITRG, and MISTRG mice (n=7-8; p-values calculated byone-way ANOVA; *, p<0.05 Tukey post hoc test). Expression was normalizedto mouse Hprt. (FIG. 11B) Flow cytometry analysis of IL-15Rα expressionon human cell populations (hCD45⁺mCD45⁻) from blood of engrafted MISTRGmice (representative of n=4). Histograms represent staining with isotypecontrol or with IL-15Rα antibody, respectively. Results arerepresentative of or combined from two experiments.

FIGS. 12A and 12B depict the results of experiments showing enhancedhuman NK cell development in MISTRG mice. (FIG. 12A and FIG. 12B)Frequency (FIG. 12A) and absolute number (FIG. 12B) of human NK cells(hNKp46⁺hCD3⁻) in engrafted NSG, MITRG, and MISTRG mice (n=8-16;p-values calculated by one-way ANOVA; *, p<0.05 Tukey post hoc test).Results are combined from four experiments.

FIGS. 13A-13F depict the results of experiments showing that bona fidehuman NK cells exhibiting enhanced maturation are present in MISTRGmice. (FIG. 13A) Flow cytometry analysis of CD94 and CD161 expression onhuman blood NK cells from a human donor and engrafted MISTRG (n=3).Histograms represent staining with isotype control Abs or withCD94/CD161 Abs. (FIG. 13B) Flow cytometry analysis of KIR expression onhuman blood NK cells from a human donor or from engrafted MISTRG mice(n=3). Numbers indicate frequencies of KIR⁺ cells. (FIG. 13C and FIG.13D) CD16 surface expression on human NK cells from engrafted NSG,MITRG, and MISTRG mice (n=4-8; p-values calculated by one-way ANOVA; *,p<0.05 Tukey post hoc test). (FIG. 13E and FIG. 13F) Intracellularperforin expression by human liver NK (hNKp46⁺hCD3⁻) and T cells (hCD3⁺)from engrafted NSG and MISTRG mice (n=3; p-value calculated by unpairedStudent's t-test). MFI, mean fluorescence intensity. Results arerepresentative of or combined from one (FIG. 13A and FIG. 13B), two(FIG. 13E and FIG. 13F), or four (FIG. 13C and FIG. 13D) experiments.

FIG. 14 depicts the results of experiments showing the effect of humanmonocyte/macrophage depletion on human NK cell homeostasis in MISTRGmice. Engrafted MISTRG mice were left untreated or treated for 3consecutive days with liposome-encapsulated clodronate to depletephagocytic cells. Flow cytometry analysis of human monocytes/macrophages(upper panel, gated on hCD33⁺ cells) and NK cells (hNKp46⁺hCD3⁻) inliver (n=8) is shown. Results are representative of two experiments. In1 out of 8 mice, the clodronate-depletion of monocytes/macrophages wasnot effective, and no reduction in NK cell number was observed in thatmouse.

FIG. 15 depicts the results of experiments showing immunohistochemistryof human myeloid cells infiltrating melanoma. Representativeimmunohistochemistry staining of human myeloid cells in tumors from NSG,MISTRG or human patients. Three subject per group, and 3 pictures persubject are shown.

FIG. 16 shows a comparison of engraftment levels and immune celldevelopment and function in recipient mice with single gene replacement,in NSG, MISTRG and in humans.

FIGS. 17A-17D depict the results of experiments demonstrating thatsamples isolated from patients with AML, CMML and MDS can be engraftedin MISTRG. (FIG. 17A) Characteristics of the samples used (includingtype of disease and genetic abnormality found in patient samples),experimental protocol (method of cell purification, number of cellsinjected per mouse and time post-transplantation at which mice wereanalyzed) and engraftment results (including number of mice withdetectable human engraftment, percentage of human hematopoietic CD45+cells and myeloid CD33+ cells, and genomic abnormality observed in humancells isolated from the mice). (FIG. 17B) Representative flow cytometryanalysis of the granularity (SSC) of myeloid CD33+ cells isolated from amouse transplanted with RAEB I patient or with normal donor cells,showing deficient granularity in RAEB I samples. (FIG. 17C)Representative fish analysis of human cells isolated from micetransplanted with RAEB II sample and showing absence of chromosome 5q.(FIG. 17D) Caryotype of human cells isolated from mice transplanted withCMML sample and showing deletion in chromosome 6.

DETAILED DESCRIPTION

The invention relates generally to a genetically modified non-humananimal expressing at least one of human M-CSF, human IL-3, human GM-CSF,human SIRPA or human TPO. The invention also relates to methods ofgenerating and methods of using the genetically modified non-humananimals described herein. In some embodiments, the genetically modifiednon-human animal is a mouse. In some embodiments, the geneticallymodified non-human animal described herein is engrafted with humanhematopoietic cells. In various embodiments, the human hematopoieticcell engrafted, genetically modified non-human animals of the inventionare useful for the in vivo evaluation of the growth and differentiationof hematopoietic and immune cells, for the in vivo evaluation of humanhematopoiesis, for the in vivo evaluation of cancer cells, for the invivo assessment of an immune response, for the in vivo evaluation ofvaccines and vaccination regimens, for the use in testing the effect ofagents that modulate cancer cell growth or survival, for the in vivoevaluation of a treatment of cancer, for the in vivo production andcollection of immune mediators, including human antibodies, and for usein testing the effect of agents that modulate hematopoietic and immunecell function.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Such terms are found definedand used in context in various standard references illustrativelyincluding J. Sambrook and D. W. Russell, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F. M.Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols;5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4thEd., Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles ofBiochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P.(Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies(scFv), heavy chain antibodies, such as camelid antibodies, andhumanized antibodies (Harlow et al., 1999, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NewYork; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “cancer” as used herein is defined as disease characterized bythe uncontrolled proliferation and/or growth of aberrant cells. Cancercells can spread locally or through the bloodstream and lymphatic systemto other parts of the body. Cancer as here herein includes both solidtumors and hematopoietic malignancies. Examples of various cancersamenable to the invention include, but are not limited to, breastcancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer,pancreatic cancer, colorectal cancer, renal cancer, liver cancer, bonecancer, brain cancer, lymphoma, leukemia, lung cancer, myeloidysplasticsyndromes, myeloproliferative disorders and the like.

“Constitutive” expression is a state in which a gene product is producedin a living cell under most or all physiological conditions of the cell.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of a mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anti-codonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding region may thusinclude nucleotide residues comprising codons for amino acid residueswhich are not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The terms “expression construct” and “expression cassette” are usedherein to refer to a double-stranded recombinant DNA molecule containinga desired nucleic acid human coding sequence and containing one or moreregulatory elements necessary or desirable for the expression of theoperably linked coding sequence.

As used herein, the term “fragment,” as applied to a nucleic acid orpolypeptide, refers to a subsequence of a larger nucleic acid orpolypeptide. A “fragment” of a nucleic acid can be at least about 15nucleotides in length; for example, at least about 50 nucleotides toabout 100 nucleotides; at least about 100 to about 500 nucleotides, atleast about 500 to about 1000 nucleotides, at least about 1000nucleotides to about 1500 nucleotides; or about 1500 nucleotides toabout 2500 nucleotides; or about 2500 nucleotides (and any integer valuein between). A “fragment” of a polypeptide can be at least about 15nucleotides in length; for example, at least about 50 amino acids toabout 100 amino acids; at least about 100 to about 500 amino acids, atleast about 500 to about 1000 amino acids, at least about 1000 aminoacids to about 1500 amino acids; or about 1500 amino acids to about 2500amino acids; or about 2500 amino acids (and any integer value inbetween).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptide.Such natural allelic variations can typically result in 1-5% variance inthe nucleotide sequence of a given gene. Alternative alleles can beidentified by sequencing the gene of interest in a number of differentindividuals. This can be readily carried out by using hybridizationprobes to identify the same genetic locus in a variety of individuals.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g. between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g. 9 of 10, arematched or homologous, the two sequences share 90% homology. By way ofexample, the DNA sequences 5′-ATTGCC-3′ and 5′-TATGGC-3′ share 50%homology.

The terms “human hematopoietic stem and progenitor cells” and “humanHSPC” as used herein, refer to human self-renewing multipotenthematopoietic stem cells and hematopoietic progenitor cells.

“Inducible” expression is a state in which a gene product is produced ina living cell in response to the presence of a signal in the cell.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

The term “operably linked” as used herein refers to a polynucleotide infunctional relationship with a second polynucleotide. By describing twopolynucleotides as “operably linked” is meant that a single-stranded ordouble-stranded nucleic acid moiety comprises the two polynucleotidesarranged within the nucleic acid moiety in such a manner that at leastone of the two polynucleotides is able to exert a physiological effectby which it is characterized, upon the other. By way of example, apromoter operably linked to the coding region of a gene is able topromote transcription of the coding region. Preferably, when the nucleicacid encoding the desired protein further comprises apromoter/regulatory sequence, the promoter/regulatory sequence ispositioned at the 5′ end of the desired protein coding sequence suchthat it drives expression of the desired protein in a cell. Together,the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof. Theterm “peptide” typically refers to short polypeptides. The term“protein” typically refers to large polypeptides.

The term “progeny” as used herein refers to a descendent or offspringand includes the differentiated or undifferentiated decedent cellderived from a parent cell. In one usage, the term progeny refers to adescendent cell which is genetically identical to the parent. In anotheruse, the term progeny refers to a descendent cell which is geneticallyand phenotypically identical to the parent. In yet another usage, theterm progeny refers to a descendent cell that has differentiated fromthe parent cell.

The term “promoter” as used herein refers to a DNA sequence operablylinked to a nucleic acid sequence to be transcribed such as a nucleicacid sequence encoding a desired molecule. A promoter is generallypositioned upstream of a nucleic acid sequence to be transcribed andprovides a site for specific binding by RNA polymerase and othertranscription factors. In specific embodiments, a promoter is generallypositioned upstream of the nucleic acid sequence transcribed to producethe desired molecule, and provides a site for specific binding by RNApolymerase and other transcription factors. An included promoter can bea constitutive promoter or can provide inducible expression; and canprovide ubiquitous, tissue-specific or cell-type specific expression.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

A “recombinant polypeptide” is one, which is produced upon expression ofa recombinant polynucleotide.

The term “regulatory element” as used herein refers to a nucleotidesequence which controls some aspect of the expression of nucleic acidsequences. Exemplary regulatory elements illustratively include anenhancer, an internal ribosome entry site (IRES), an intron; an originof replication, a polyadenylation signal (pA), a promoter, an enhancer,a transcription termination sequence, and an upstream regulatory domain,which contribute to the replication, transcription, post-transcriptionalprocessing of a nucleic acid sequence. Those of ordinary skill in theart are capable of selecting and using these and other regulatoryelements in an expression construct with no more than routineexperimentation. Expression constructs can be generated recombinantly orsynthetically using well-known methodology.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specificallybinding”, can be used in reference to the interaction of an antibody, aprotein, or a peptide with a second chemical species, to mean that theinteraction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the chemical species; forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally. If an antibody is specificfor epitope “A”, the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialbiological properties of the reference molecule. Changes in the sequenceof a nucleic acid variant may not alter the amino acid sequence of apeptide encoded by the reference nucleic acid, or may result in aminoacid substitutions, additions, deletions, fusions and truncations.Changes in the sequence of peptide variants are typically limited orconservative, so that the sequences of the reference peptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference peptide can differ in amino acid sequence by oneor more substitutions, additions, deletions in any combination. Avariant of a nucleic acid or peptide can be a naturally occurring suchas an allelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

As used herein, the term “genetically modified” means an animal, thegerm cells of which comprise an exogenous human nucleic acid or humannucleic acid sequence. By way of non-limiting examples a geneticallymodified animal can be a transgenic animal or a knock-in animal, so longas the animal comprises a human nucleic acid sequence.

As used herein, “knock-in” is meant a genetic modification that replacesthe genetic information encoded at a chromosomal locus in a non-humananimal with a different DNA sequence.

Description

The invention relates to a genetically modified non-human animalexpressing human M-CSF, human IL-3/GM-CSF, human SIRPA and human TPO(herein referred to as MIST). The invention also relates to methods ofgenerating and methods of using the genetically modified non-humananimals described herein. In some embodiments, the genetically modifiednon-human animal is a mouse. In some embodiments, the geneticallymodified non-human animal is an immunodeficient mouse. In a particularembodiment, the immunodeficient mouse is a RAG2^(−/−) γ_(c) ^(−/−)mouse. In another particular embodiment, the genetically modifiednon-human animal of the invention expresses human M-CSF, humanIL-3/GM-CSF, and human TPO and does not express RAG2 or γ_(c) (referredto herein as MITRG). In another particular embodiment, the geneticallymodified non-human animal of the invention expresses human M-CSF, humanIL-3/GM-CSF, human SIRPA and human TPO and does not express RAG2 orγ_(c) (referred to herein as MISTRG). In some embodiments, thegenetically modified non-human animals described herein are engraftedwith a human hematopoietic cell.

In various embodiments, the human hematopoietic cell engrafted,genetically modified non-human animals of the invention are useful forthe in vivo evaluation of the growth and differentiation ofhematopoietic and immune cells, for the in vivo evaluation of humanhematopoiesis, for the in vivo evaluation of cancer cells, for the invivo assessment of an immune response, for the in vivo evaluation ofvaccines and vaccination regimens, for the use in testing the effect ofagents that modulate cancer cell growth or survival, for the in vivoevaluation of a treatment of cancer, for the in vivo production andcollection of immune mediators, including human antibodies, and for usein testing the effect of agents that modulate hematopoietic and immunecell function.

Genetically Modified Non-Human Animals

The invention includes a genetically modified non-human animal thatexpresses at least one of human M-CSF, human IL-3/GM-CSF, human SIRPA,human TPO, and any combination thereof. In some embodiments, thegenetically modified non-human animal that expresses a human nucleicacid also expresses the corresponding non-human animal nucleic acid. Inother embodiments, the genetically modified non-human animal thatexpresses a human nucleic acid does not also express the correspondingnon-human animal nucleic acid. In some embodiments, the geneticallymodified animal is an animal having one or more genes knocked out torender the animal an immunodeficient animal, as elsewhere describedherein. To create a genetically modified non-human animal, a nucleicacid encoding a human protein can be incorporated into a recombinantexpression vector in a form suitable for expression of the human proteinin a non-human host cell. In various embodiments, the recombinantexpression vector includes one or more regulatory sequences operativelylinked to the nucleic acid encoding the human protein in a manner whichallows for transcription of the nucleic acid into mRNA and translationof the mRNA into the human protein. The term “regulatory sequence” isart-recognized and intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are known to those skilled in the art and aredescribed in 1990, Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. It should beunderstood that the design of the expression vector may depend on suchfactors as the choice of the host cell to be transfected and/or theamount of human protein to be expressed.

A genetically modified animal can be created, for example, byintroducing a nucleic acid encoding the human protein (typically linkedto appropriate regulatory elements, such as a constitutive ortissue-specific enhancer) into an oocyte, e.g., by microinjection, andallowing the oocyte to develop in a female foster animal. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene.Methods for generating genetically modified animals, particularlyanimals such as mice, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009 and1986, Hogan et al., A Laboratory Manual, Cold Spring Harbor, N.Y., ColdSpring Harbor Laboratory. A genetically modified founder animal can beused to breed additional animals carrying the transgene. Geneticallymodified animals carrying a transgene encoding the human protein of theinvention can further be bred to other genetically modified animalscarrying other transgenes, or be bred to knockout animals, e.g., aknockout animal that does not express one or more of its genes. Invarious embodiments, the genetically modified animal of the invention isa mouse, a rat or a rabbit.

In some embodiments, the genetically modified animal of the inventionexpresses one or more human nucleic acids from the non-human animal'snative promoter and native regulatory elements. In other embodiments,the genetically modified animal of the invention expresses a humannucleic acid from the native human promoter and native regulatoryelements. The skilled artisan will understand that the geneticallymodified animal of the invention includes genetically modified animalsthat express at least one human nucleic acid from any promoter. Examplesof promoters useful in the invention include, but are not limited to,DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter,globin promoter, ovalbumin promoter, SV40 early promoter, the Roussarcoma virus (RSV) promoter, retroviral LTR and lentiviral LTR.Promoter and enhancer expression systems useful in the invention alsoinclude inducible and/or tissue-specific expression systems.

In some embodiments, the invention includes genetically modifiedimmunodeficient animals having a genome that includes a nucleic acidencoding a human polypeptide operably linked to a promoter, wherein theanimal expresses the encoded human polypeptide. In various embodiments,the invention includes genetically modified immunodeficient non-humananimals having a genome that comprises an expression cassette thatincludes a nucleic acid encoding at least one human polypeptide, whereinthe nucleic acid is operably linked to a promoter and a polyadenylationsignal and further contains an intron, and wherein the animal expressesthe encoded human polypeptide.

In various embodiments, various methods are used to introduce a humannucleic acid sequence into an immunodeficient animal to produce agenetically modified immunodeficient animal that expresses a human gene.Such techniques are well-known in the art and include, but are notlimited to, pronuclear microinjection, transformation of embryonic stemcells, homologous recombination and knock-in techniques. Methods forgenerating genetically modified animals that can be used include, butare not limited to, those described in Sundberg and Ichiki (2006,Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen(2002, Genetically modified Mouse Methods and Protocols, Humana Press),Joyner (2000, Gene Targeting: A Practical Approach, Oxford UniversityPress), Turksen (2002, Embryonic stem cells: Methods and Protocols inMethods Mol Biol., Humana Press), Meyer et al. (2010, Proc. Nat. Acad.Sci. USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science2^(nd) ed. Sunderland, Mass.: Sinauer), U.S. Pat. No. 6,586,251,Rathinam et al. (2011, Blood 118:3119-28), Willinger et al., (2011, ProcNatl Acad Sci USA, 108:2390-2395), Rongvaux et al., (2011, Proc NatlAcad Sci USA, 108:2378-83) and Valenzuela et al. (2003, Nat Biot21:652-659).

In some embodiments, the compositions and methods of the inventioncomprise genetically modified immunodeficient animals deficient in Bcell and/or T cell number and/or function, alone, or in combination witha deficiency in NK cell number and/or function (for example, due to anIL2 receptor gamma chain deficiency (i.e., γ_(c) ^(−/−))), and having agenome that comprises a human nucleic acid operably linked to apromoter, wherein the animal expresses the encoded human polypeptide.The generation of the genetically modified animal of the invention canbe achieved by methods such as DNA injection of an expression constructinto a preimplantation embryo or by use of stem cells, such as embryonicstem (ES) cells or induced pluripotent stem (iPS) cells.

In one embodiment, the human nucleic acid is expressed by the nativeregulatory elements of the human gene. In other embodiments, the humannucleic acid is expressed by the native regulatory elements of thenon-human animal. In other embodiments, human nucleic acid is expressedfrom a ubiquitous promoter. Nonlimiting examples of ubiquitous promotersuseful in the expression construct of the compositions and methods ofthe invention include, a 3-phosphoglycerate kinase (PGK-1) promoter, abeta-actin promoter, a ROSA26 promoter, a heat shock protein 70 (Hsp70)promoter, an EF-1 alpha gene encoding elongation factor 1 alpha (EF1)promoter, an eukaryotic initiation factor 4A (eIF-4A1) promoter, achloramphenicol acetyltransferase (CAT) promoter and a CMV(cytomegalovirus) promoter.

In other embodiments, the human nucleic acid is expressed from atissue-specific promoter. Nonlimiting examples of tissue-specificpromoters useful in the expression construct of the compositions andmethods of the invention include a promoter of a gene expressed in thehematopoietic system, such as a M-CSF promoter, an IL-3 promoter, aGM-CSF promoter, a SIRPA promoter, a TPO promoter, an IFN-β promoter, aWiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also calledleukocyte common antigen) promoter, a Flt-1 promoter, an endoglin(CD105) promoter and an ICAM-2 (Intracellular Adhesion Molecule 2)promoter. These and other promoters useful in the compositions andmethods of the invention are known in the art as exemplified in Abboudet al. (2003, J. Histochem & Cytochem. 51:941-949), Schorpp et al.(1996, NAR 24:1787-1788), McBurney et al. (1994, Devel. Dynamics,200:278-293) and Majumder et al. (1996, Blood 87:3203-3211). Further tocomprising a promoter, one or more additional regulatory elements, suchas an enhancer element or intron sequence, is included in variousembodiments of the invention. Examples of enhancers useful in thecompositions and methods of the invention include, but are not limitedto, a cytomegalovirus (CMV) early enhancer element and an SV40 enhancerelement. Examples of intron sequences useful in the compositions andmethods of the invention include, but are not limited to, the betaglobin intron or a generic intron. Other additional regulatory elementsuseful in some embodiments of the invention include, but are not limitedto, a transcription termination sequence and an mRNA polyadenylation(pA) sequence.

In some embodiments, the methods of introduction of the human nucleicacid expression construct into a preimplantation embryo includelinearization of the expression construct before it is injected into apreimplantation embryo. In preferred embodiments, the expressionconstruct is injected into fertilized oocytes. Fertilized oocytes can becollected from superovulated females the day after mating and injectedwith the expression construct. The injected oocytes are either culturedovernight or transferred directly into oviducts of 0.5-day p.c.pseudopregnant females. Methods for superovulation, harvesting ofoocytes, expression construct injection and embryo transfer are known inthe art and described in Manipulating the Mouse Embryo (2002, ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press).Offspring can be evaluated for the presence of the introduced nucleicacid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) orby protein analysis (e.g., ELISA, Western blot, etc.).

In other embodiments, the expression construct may be transfected intostem cells (ES cells or iPS cells) using well-known methods, such aselectroporation, calcium-phosphate precipitation and lipofection. Thecells can be evaluated for the presence of the introduced nucleic acidby DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or byprotein analysis (e.g., ELISA, Western blot, etc.). Cells determined tohave incorporated the expression construct can then be microinjectedinto preimplantation embryos. For a detailed description of methodsknown in the art useful for the compositions and methods of theinvention, see Nagy et al., (2002, Manipulating the Mouse Embryo: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press),Nagy et al. (1990, Development 110:815-821), U.S. Pat. No. 7,576,259,U.S. Pat. No. 7,659,442, U.S. Pat. No. 7,294,754, and Kraus et al.(2010, Genesis 48:394-399).

The genetically modified non-human animals of the invention can becrossed to immunodeficient animal to create an immunodeficient animalexpressing at least one human nucleic acid. Various embodiments of theinvention provide genetically modified animals that include a humannucleic acid in substantially all of their cells, as well as geneticallymodified animals that include a human nucleic acid in some, but not alltheir cells. One or multiple copies, adjacent or distant to one another,of the human nucleic acid may be integrated into the genome of the cellsof the genetically modified animals.

In some embodiments, the invention is a genetically modified non-humanmouse engrafted with at least one human hematopoietic cell. In otherembodiments, the invention is a method of engrafting human hematopoieticcells in a genetically modified non-human animal. The engrafted humanhematopoietic cells useful in the compositions and methods of theinvention include any human hematopoietic cell. Non-limiting examples ofhuman hematopoietic cells useful in the invention include, but are notlimited to, HSC, HSPC, leukemia initiating cells (LIC), andhematopoietic cells of any lineage at any stage of differentiation,including terminally differentiated hematopoietic cells of any lineage.Such hematopoietic cells can be derived from any tissue or location of ahuman donor, including, but not limited to, bone marrow, peripheralblood, liver, fetal liver, or umbilical cord blood. Such hematopoieticcells can be isolated from any human donor, including healthy donors, aswell as donors with disease, such as cancer, including leukemia.

In other embodiments, the invention is a method of engrafting humanhematopoietic cells in a genetically modified non-human animal. In someembodiments, the genetically modified non-human animal into which humanhematopoietic cells are engrafted is an immunodeficient animal.Engraftment of hematopoietic cells in the genetically modified animal ofthe invention is characterized by the presence of human hematopoieticcells in the engrafted animal. In particular embodiments, engraftment ofhematopoietic cells in an immunodeficient animal is characterized by thepresence of differentiated human hematopoietic cells in the engraftedanimal in which hematopoietic cells are provided, as compared withappropriate control animals.

In some embodiments, the animals of the invention are transplanted withhuman cancer cells (e.g., human solid tumors, etc.) in addition to humanhematopoietic cells. In various embodiments, the human cancer cells canbe a cancer cell line or primary human cancer cell isolated from apatient, from any of many different types of cancer (including, by wayof non-limiting examples, melanoma, breast cancer, lung cancer, etc.) Insome embodiments, the human cancer cell and the HSPC are isolated fromthe same patient and transplanted into the same non-human animal.

The genetically modified non-human animals provided in variousembodiments of the present invention have various utilities such as, butnot limited to, for use as models of growth and differentiation ofhematopoietic cells, for the in vivo evaluation of human hematopoiesis,for the in vivo evaluation of cancer cells, for in vivo study of animmune response, for in vivo evaluation of vaccines and vaccinationregimens, for the use in testing the effect of agents that modulatecancer cell growth or survival, for the in vivo evaluation of atreatment of cancer, for in vivo production and collection of immunemediators, such as an antibody, and for use in testing the effect ofagents that affect hematopoietic and immune cell function.

Engraftment of human hematopoietic cells in genetically modified and/orimmunodeficient non-human animals has traditionally requiredconditioning prior to administration of the hematopoietic cells, eithersub-lethal irradiation of the recipient animal with high frequencyelectromagnetic radiation, generally using gamma or X-ray radiation, ortreatment with a radiomimetic drug such as busulfan or nitrogen mustard.Conditioning is believed to reduce numbers of host hematopoietic cells,create appropriate microenvironmental factors for engraftment of humanhematopoietic cells, and/or create microenvironmental niches forengraftment of human hematopoietic cells. Standard methods forconditioning are known in the art, such as described herein and in J.Hayakawa et al, 2009, Stem Cells, 27(1):175-182. Methods for engraftmentof human hematopoietic cells in immunodeficient animals are providedaccording to embodiments of the present invention which includeproviding human hematopoietic cells to the immunodeficient animals, withor without irradiating the animals prior to administration of thehematopoietic cells. Methods for engraftment of human hematopoieticcells in immunodeficient animals are provided according to embodimentsof the present invention which include providing human hematopoieticcells to the genetically modified non-human animals of the invention,with or without, administering a radiomimetic drug, such as busulfan ornitrogen mustard, to the animals prior to administration of thehematopoietic cells.

In some embodiments, the methods of hematopoietic cell engraftment in agenetically modified non-human animal according to embodiments of thepresent invention include providing human hematopoietic cells to agenetically modified animal of the invention as elsewhere describedhere. In some embodiments, the genetically modified non-human animal ofthe invention is an immunodeficient animal that is deficient innon-human B cell number and/or function, non-human T cell number and/orfunction, and/or non-human NK cell number and/or function. In otherembodiments, the immunodeficient animal has severe combined immunedeficiency (SCID). SCID refers to a condition characterized by theabsence of T cells and lack of B cell function. Examples of SCIDinclude: X-linked SCID, which is characterized by gamma chain genemutations in the IL2RG gene and the lymphocyte phenotype T(−) B(+)NK(−); and autosomal recessive SCID characterized by Jak3 gene mutationsand the lymphocyte phenotype T(−) B(+) NK(−), ADA gene mutations and thelymphocyte phenotype T(−) B(−) NK(−), IL-7R alpha-chain mutations andthe lymphocyte phenotype T(−) B(+) NK(+), CD3 delta or epsilon mutationsand the lymphocyte phenotype T(−) B(+) NK(+), RAG1/RAG2 mutations andthe lymphocyte phenotype T(−) B(−) NK(+), Artemis gene mutations and thelymphocyte phenotype T(−) B(−) NK(+), CD45 gene mutations and thelymphocyte phenotype T(−) B(+) NK(+). In some embodiments, thegenetically modified non-human animal of the invention is RAG1^(−/−).

In some embodiments, the methods of hematopoietic cell engraftment in agenetically modified animal according to embodiments of the presentinvention include providing human hematopoietic cell to in a geneticallymodified non-human animal having the severe combined immunodeficiencymutation (Prkdc^(scid)), commonly referred to as the scid mutation. Thescid mutation is well-known and located on mouse chromosome 16 asdescribed in Bosma et al. (1989, Immunogenetics 29:54-56). Micehomozygous for the scid mutation are characterized by an absence offunctional T cells and B cells, lymphopenia, hypoglobulinemia and anormal hematopoietic microenvironment. The scid mutation can bedetected, for example, by detection of markers of the scid mutationusing well-known methods.

In other embodiments, the methods of hematopoietic cell engraftment in agenetically modified animal according to embodiments of the presentinvention include providing human hematopoietic cells to geneticallymodified immunodeficient non-human animal having an IL2 receptor gammachain deficiency, either alone, or in combination with, the severecombined immunodeficiency (scid) mutation. The term “IL2 receptor gammachain deficiency” refers to decreased IL2 receptor gamma chain.Decreased IL2 receptor gamma chain can be due to gene deletion ormutation. Decreased IL2 receptor gamma chain can be detected, forexample, by detection of IL2 receptor gamma chain gene deletion ormutation and/or detection of decreased IL2 receptor gamma chainexpression using well-known methods.

In addition to the naturally occurring human nucleic acid and amino acidsequences, the term encompasses variants of human nucleic acid and aminoacid sequences As used herein, the term “variant” defines either anisolated naturally occurring genetic mutant of a human or arecombinantly prepared variation of a human, each of which contain oneor more mutations compared with the corresponding wild-type human. Forexample, such mutations can be one or more amino acid substitutions,additions, and/or deletions. The term “variant” also includes non-humanorthologues. In some embodiments, a variant polypeptide of the presentinvention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to a wild-type human polypeptide.

The percent identity between two sequences is determined usingtechniques as those described elsewhere herein. Mutations can beintroduced using standard molecular biology techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. One of skill inthe art will recognize that one or more amino acid mutations can beintroduced without altering the functional properties of human proteins.

Conservative amino acid substitutions can be made in human proteins toproduce human protein variants. Conservative amino acid substitutionsare art recognized substitutions of one amino acid for another aminoacid having similar characteristics. For example, each amino acid may bedescribed as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate, glutamate; basicamino acids include histidine, lysine, arginine; aliphatic amino acidsinclude isoleucine, leucine and valine; aromatic amino acids includephenylalanine, glycine, tyrosine and tryptophan; polar amino acidsinclude aspartate, glutamate, histidine, lysine, asparagine, glutamine,arginine, serine, threonine and tyrosine; and hydrophobic amino acidsinclude alanine, cysteine, phenylalanine, glycine, isoleucine, leucine,methionine, proline, valine and tryptophan; and conservativesubstitutions include substitution among amino acids within each group.Amino acids may also be described in terms of relative size, alanine,cysteine, aspartate, glycine, asparagine, proline, threonine, serine,valine, all typically considered to be small.

Human variants can include synthetic amino acid analogs, amino acidderivatives and/or non-standard amino acids, illustratively including,without limitation, alpha-aminobutyric acid, citrulline, canavanine,cyanoalanine, diaminobutyric acid, diaminopimelic acid,dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline,norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan,1-methylhistidine, methylhistidine, and ornithine.

Human variants are encoded by nucleic acids having a high degree ofidentity with a nucleic acid encoding a wild-type human. The complementof a nucleic acid encoding a human variant specifically hybridizes witha nucleic acid encoding a wild-type human under high stringencyconditions.

The term “nucleic acid” refers to RNA or DNA molecules having more thanone nucleotide in any form including single-stranded, double-stranded,oligonucleotide or polynucleotide. The term “nucleotide sequence” refersto the ordering of nucleotides in an oligonucleotide or polynucleotidein a single-stranded form of nucleic acid.

Nucleic acids encoding a human variant can be isolated or generatedrecombinantly or synthetically using well-known methodology.

Isolation of human hematopoietic cells, administration of the humanhematopoietic cells to a host animal and methods for assessingengraftment thereof are well-known in the art. Hematopoietic cells foradministration to host animal can be obtained from any tissue containinghematopoietic cells such as, but not limited to, umbilical cord blood,bone marrow, peripheral blood, cytokine or chemotherapy-mobilizedperipheral blood and fetal liver. Hematopoietic cells can beadministered into newborn or adult animals by administration via variousroutes, such as, but not limited to, intravenous, intrahepatic,intraperitoneal, intrafemoral and/or intratibial.

Engraftment of human hematopoietic cells in the genetically modifiedanimal of the invention can be assessed by any of various methods, suchas, but not limited to, flow cytometric analysis of cells in the animalsto which the human hematopoietic cells are administered at one or moretime points following the administration of hematopoietic cells.

Exemplary methods of isolating human hematopoietic cells, ofadministering human hematopoietic cells to a host animal, and ofassessing engraftment of the human hematopoietic cells in the hostanimal are described herein and in Pearson et al. (2008, Curr. Protoc.Immunol. 81:1-15), Ito et al. (2002, Blood 100:3175-3182), Traggiai etal. (2004, Science 304:104-107), Ishikawa et al. (2005, Blood106:1565-1573), Shultz et al. (2005, J. Immunol. 174:6477-6489) andHolyoake et al. (1999, Exp Hematol. 27:1418-27).

In some embodiments of the invention, the human hematopoietic cells areisolated from an original source material to obtain a population ofcells enriched for a particular hematopoietic cell population (e.g.,HSCs, HSPCs, LICs, CD34+, CD34−, lineage specific marker, etc.). Theisolated hematopoietic cells may or may not be a pure population. In oneembodiment, hematopoietic cells useful in the compositions and methodsof the invention are depleted of cells having a particular marker, suchas CD34. In another embodiment, hematopoietic cells useful in thecompositions and methods of the invention are enriched by selection fora marker, such as CD34. In some embodiments, hematopoietic cells usefulin the compositions and methods of the invention are a population ofcells in which CD34+ cells constitute about 1-100% of the cells,although in certain embodiments, a population of cells in which CD34+cells constitute fewer than 1% of total cells can also be used. Incertain embodiments, the hematopoietic cells useful in the compositionsand methods of the invention are a T cell-depleted population of cellsin which CD34+ cells make up about 1-3% of total cells, alineage-depleted population of cells in which CD34+ cells make up about50% of total cells, or a CD34+ positive selected population of cells inwhich CD34+ cells make up about 90% of total cells.

The number of hematopoietic cells administered is not consideredlimiting with regard to the generation of a human hematopoietic and/orimmune system in a genetically modified non-human animal expressing atleast one human gene. Thus, by way of non-limiting example, the numberof hematopoietic cells administered can range from about 1×10³ to about1×10⁷, although in various embodiments, more or fewer can also be used.By way of another non-limiting example, the number of HSPCs administeredcan range from about 3×10³ to about 1×10⁶ CD34+ cells when the recipientis a mouse, although in various embodiments, more or fewer can also beused. For other species of recipient, the number of cells that need tobe administered can be determined using only routine experimentation.

Generally, engraftment can be considered successful when the number (orpercentage) of human hematopoietic cells present in the geneticallymodified non-human animal is greater than the number (or percentage) ofhuman cells that were administered to the non-human animal, at a pointin time beyond the lifespan of the administered human hematopoieticcells. Detection of the progeny of the administered hematopoietic cellscan be achieved by detection of human DNA in the recipient animal, forexample, or by detection of intact human hematopoietic cells, such as bythe detection of the human cell surface marker, such as CD45 forexample. Serial transfer of human hematopoietic cells from a firstrecipient into a secondary recipient, and engraftment of humanhematopoietic cells in the second recipient, is a further optional testof engraftment in the primary recipient. Engraftment can be detected byflow cytometry as 0.05% or greater human CD45+ cells in the blood,spleen or bone marrow at 1-4 months after administration of the humanhematopoietic cells. A cytokine (e.g., GM-CSF) can be used to mobilizestem cells, for example, as described in Watanabe (1997, Bone MarrowTransplantation 19:1175-1181).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Functional Innate Immune Responses and Solid Tumor Support inHuman-Hemato-Lymphoid System Mice

As described herein, mice repopulated with a human hemato-lymphoidsystem (HHLS) represent a powerful tool for predictive human preclinicalin vivo research. A major limitation of current HHLS mice is thedefective development of human cells critical for innate immunity. Here,a novel mouse strain is reported in which multiple genes encodingcytokines are genetically humanized. These humanized cytokines actsynergistically to efficiently support human hematopoiesis and thedevelopment and function of human monocytes/macrophages and NK cells. Ina tumor microenvironment, human macrophages acquire an immunosuppressivephenotype and support the growth of a human cancer. With a more completeand functional human innate immune system, this novel model of HHLS micehas exceptional potential to facilitate the study of physiology andpathology of human innate immunity in vivo.

Monocytes and macrophages are major cellular components of the innateimmune response (Auffray et al., 2009, Annual review of immunology 27,669). On the one hand, these cells are capable of sensing an infectionand of mediating direct anti-microbial functions, by diverse mechanismssuch as phagocytosis or the secretion of pro-inflammatory factors. Onthe other hand, monocytes/macrophages can acquire immunosuppressivefunctions, important for the resolution of inflammation and for tissuerepair. Furthermore, these anti-inflammatory properties can be co-optedby tumor-infiltrating macrophages and provide a survival advantage toevolving tumors through a diversity of mechanisms (Allavena andMantovani, 2012, Clinical and experimental immunology 167, 195; Qian andPollard, 2010, Cell 141, 39).

Small animal models such as mice are frequently used to study in vivomammalian immune responses. However, fundamental differences in immunefunction exist between species (Mestas and Hughes, 2004, Journal ofImmunology 172, 2731; Rongvaux et al., 2013, Annual review of immunology31, 635). In particular, major phenotypic and functionalspecies-specific differences exist among monocytes/macrophagespopulations and generally, knowledge gained from mouse studies is onlypartly applicable to humans (Auffray et al., 2009, Annual review ofimmunology 27, 669; Rongvaux et al., 2013, Annual review of immunology31, 635; Chow et al., 2011, Nature reviews Immunology 11, 788). Onepromising approach to study the specificities of human hematopoietic andimmune function in vivo consists in using mice carrying a humanhemato-lymphoid system (HHLS) (Rongvaux et al., 2013, Annual review ofimmunology 31, 635; Shultz et al., 2012, Nature reviews Immunology 12,786). However, the development and function of several human immune celltypes, such as monocytes/macrophages and NK cells, is largely defectivein current HHLS mice (Rongvaux et al., 2013, Annual review of immunology31, 635). These defects are most likely due to reduced cross-reactivityof mouse cytokines on the corresponding human receptors (Manz, 2007,Immunity 26, 537). To circumvent this limitation, a strategy wasdeveloped to replace mouse genes encoding cytokines by their humancounterpart (Willinger et al., 2011, Trends in immunology 32, 321) andthis approach resulted in significant improvements in the developmentand function of individual human cell types (FIG. 16) (Rathinam et al.,2011, Blood 118, 3119; Willinger et al., 2011, Proceedings of theNational Academy of Sciences 108, 2390; Rongvaux et al., 2011,Proceedings of the National Academy of Sciences 108, 2378).

Hematopoiesis is a tightly regulated developmental process in whichmultipotent hematopoietic stem cells differentiate into more committedprogenitors and then into mature blood cells (Kondo et al., 2003, Annualreview of immunology 21, 759; Doulatov et al., 2012, Cell stem cell 10,120). This process requires specific cytokines that support successivedevelopmental steps (FIG. 5). Perhaps synergy between multiple humanizedcytokines would be required to fully recapitulate human myelopoiesis inthe mouse. Thus, a novel mouse strain, named MISTRG, was generated inwhich the genes encoding M-CSF (Rathinam et al., 2011, Blood 118, 3119),IL-3/GM-CSF (Willinger et al., 2011, Proceedings of the National Academyof Sciences 108, 2390) and TPO (Rongvaux et al., 2011, Proceedings ofthe National Academy of Sciences 108, 2378) were replaced by their humancounterparts (Willinger et al., 2011, Trends in immunology 32, 321) inthe hSIRPAtg RAG2−/− IL-2Rγ−/− background (Traggiai et al., 2004,Science 304, 104; Strowig et al., 2011, Proceedings of the NationalAcademy of Sciences 108, 13218).

Newborn MISTRG mice and their littermates MITRG (lacking the hSIRPAtransgene) were sublethally irradiated and transplanted with human fetalliver-derived CD34+ cells, following a standard protocol (Traggiai etal., 2004, Science 304, 104). RAG2−/− IL2-Rγ−/− (RG) mice that share thesame genetic background but lack all the humanized alleles, andcommercially available NOD-Scid IL2-Rγ−/− (NSG) mice served as controls.Blood engraftment levels (hCD45+ cell percentage; (FIGS. 1A and 1B; andFIG. 6A) were lower in RG and higher in NSG recipients as previouslyreported (Strowig et al., 2011, Proceedings of the National Academy ofSciences 108, 13218; Brehm et al., 2010, Clinical immunology 135, 84).The percentage of blood hCD45+ cells was similar in MISTRG and in NSG.Blood engraftment was also significantly increased in MITRG compared toRG, suggesting that the combined humanization of genes overcomes theneed to induce phagocytic tolerance through SIRPα/CD47 cross-reactivity(Strowig et al., 2011, Proceedings of the National Academy of Sciences108, 13218; Takenaka et al., 2007, Nature immunology 8, 1313; Legrand etal., 2011, Proceedings of the National Academy of Sciences 108, 13224),possibly by weakening the mouse innate response. Mice with at least 10%human CD45+ cells in the blood were selected for further experimentation(FIG. 6B). In the bone marrow (BM), the percentages of hCD45+ cellsexceeded 90% and reached up to 99% in the majority of both MISTRGrecipients (FIGS. 1A and 1C; and FIGS. 6C to 6E), and the highefficiency of engraftment in the BM was independent of SIRPα/CD47interaction. To test the capacity of humanized cytokines to supporthuman hematopoiesis in more competitive conditions, human CD34+ cellswere transplanted into non-irradiated MISTRG. This protocol resulted inhuman CD45+ cells in the blood and BM of all recipients (FIGS. 1D and1E) and remarkably, half of the mice showed chimerism as high as thehighest levels measured in recipients engrafted after X-raypre-conditioning (compare FIG. 1E to FIGS. 1B and 1C). The datadescribed herein show that the genetic replacement of multiple cytokinesin MISTRG creates a microenvironment in which human hematopoiesis canalmost completely displace mouse hematopoiesis in the bone marrow, andobviate the need for pathology-inducing irradiation.

Next, the capacity of MISTRG mice to support human myelopoiesis wasassessed. Human myeloid cells (hCD33+) were present in significantlyhigher proportions in the blood and bone marrow of MISTRG compared to RGand NSG (FIG. 2A; and FIGS. 7A to 7C). The increased proportion ofmyeloid cells in MISTRG resulted in a blood composition that resemblesthe physiological composition of human blood, which is rich in myeloidcells and radically different from that of lymphoid-rich mouse blood(Mestas and Hughes, 2004, Journal of Immunology 172, 2731; Rongvaux etal., 2013, Annual review of immunology 31, 6354) (FIG. 2B; and FIG. 7D).While both monocytes (CD33hiSSCloCD66−) and granulocytes(CD33+SSChiCD66+) were present in the BM (FIG. 7E), human myeloid cellpopulations in peripheral blood were composed mostly of monocytes (FIG.7F), suggesting that the terminal differentiation and egress from the BMor peripheral survival of human granulocytes is still suboptimal in thismouse environment. Importantly however, human myeloid cells were presentin high numbers in non-lymphoid tissues such as lung, liver and colon ofMISTRG as shown by immunohistochemistry (hCD68+ cells; (FIG. 2C) or byflow cytometry (hCD33+; (FIGS. 7G and 7H), and significantly exceededhuman myeloid cell numbers found in NSG mice by a factor of ˜10.

In humans, three subsets of monocytes have been phenotypically andfunctionally described, based on the expression of the CD14 and CD16markers (Auffray et al., 2009, Annual review of immunology 27, 669; Croset al., 2010, Immunity 33, 375). All three subpopulations of humanmonocytes (CD14+CD16−, CD14+CD16+ and CD14dimCD16+) were present in thelymphoid and non-lymphoid tissues, such as lung and liver, of MISTRG(FIGS. 2D and 2E; and FIGS. 8A and 8B). In contrast in NSG, in additionto the lower frequency of myeloid cells, only CD14+CD16− and to someextent CD14+CD16+ monocytes could be consistently detected, whileCD14dimCD16+ cells were only marginally represented. The extendedimmunophenotype (CD33, CD11b, CD115, CD62L and CX3CR1) of the monocytesubpopulations found in MISTRG compared closely to the equivalentsubsets in human peripheral blood (FIGS. 9A and 9B). Human CD14+CD16-and CD14+CD16+ monocytes isolated from the BM of MITRG produced highlevels of inflammatory cytokines in response to TLR4 and TLR7/8 ligands(LPS and R848, respectively) (FIGS. 2F and 2G). In an in vitro assayperformed on WBCs of MITRG, both CD14+CD16- and CD14+CD16+ cells had ahigh capacity to phagocytose GFP-expressing E. coli, while CD14dimCD16+monocytes had limited phagocytic ability (FIG. 2H), again reflecting thephysiological properties of the corresponding subpopulations in humanblood (Cros et al., 2010, Immunity 33, 375). When challenged in vivowith LPS or infected with the bacterial and viral human pathogensListeria monocytogenes and influenza A, respectively, MISTRG miceresponded with robust production of human inflammatory cytokines (TNFα,IL-6 and IFNγ, respectively), while NSG mice showed significantly lower,about one log lower, responses (FIGS. 2I to 2K). These resultsdemonstrate that the human monocyte subsets that develop in MISTRG arefunctional in vitro and in vivo. However, a drawback of the presence offunctional human phagocytic cells in the mouse is a breach ofhuman-to-mouse phagocytic tolerance, to which mouse RBCs areparticularly susceptible (FIGS. 10A and 10B). This destruction of mouseRBCs resulted in anemia (FIGS. 10C to 10I) and limited the lifespan ofengrafted mice to 10-12 weeks (MISTRG) or 12-16 weeks (MITRG).

Myeloid cells can support the development and differentiation of otherimmune cells through the production of cytokines. Whether the myeloidcompartment of MISTRG mice was a source of human cytokines, such asIL-15, was assessed. Consistent with this notion, it was found that mRNAexpression of human IL-15 and IL-15Rα was increased by a factor ofgreater than 10 in MISTRG when compared to NSG (FIG. 3A; and FIG. 11A).To define in more detail the cellular source of human IL-15/IL-15Rα inMISTRG, the abundance of human IL-15 and IL-15Rα transcripts in purifiedhuman cell populations was measured. Expression of human IL-15Rα mRNAwas higher in human myeloid cells (hCD33+) than in non-myeloid cells(hCD33−) (FIG. 3B). In particular, CD14+CD16+ monocytes showed anenrichment of both IL-15 and IL-15Rα transcripts (FIG. 3B). Theexpression of human IL-15Rα protein on the surface of human myeloidcells from MISTRG was confirmed by flow cytometry (FIG. 11B).

Based on these findings, whether MISTRG mice support the development ofhuman immune cells dependent on IL-15 trans-presentation, such as NKcells (Ma et al., 2006, Annual review of immunology 24, 657; Soderquestet al., 2011, Blood 117, 4511), was assessed. The efficient developmentof human NK cells in current HHLS mouse models requires the exogenouspharmacologic delivery of human IL-15/IL-15Rα (Huntington et al., 2009,Journal of experimental medicine 206, 25; Chen et al., 2009, Proceedingsof the National Academy of Sciences 106, 21783; Pek et al., 2011,Immunobiology 216, 218)23-25) since mouse IL-15 is not sufficient tosupport human NK cells in vivo. As previously reported (Huntington etal., 2009, Journal of experimental medicine 206, 25; Chen et al., 2009,Proceedings of the National Academy of Sciences 106, 21783; Pek et al.,2011, Immunobiology 216, 218), very few human NK cells (hNKp46+hCD3−)were observed in engrafted NSG (FIGS. 3C and 3D; and FIGS. 12A and 12B).In contrast, human NK cells were readily detected in multiple tissues ofengrafted MISTRG and were increased by a factor of ˜10 compared to NSG(FIGS. 3C and 3D; and FIGS. 12A and 12B). Apart from the bone marrow,MITRG had less human NK cells than MISTRG, which is most likely due tothe previously reported requirement for human SIRPα for the survival ofhuman NK cells in the periphery (Legrand et al., 2011, Proceedings ofthe National Academy of Sciences 108, 13224). The hNKp46+hCD3− cells inMISTRG mice represented bona fide NK cells because they expressed thetypical NK cell surface markers CD94, CD161, and killer inhibitoryreceptors (KIRs) closely mimicking human controls (FIGS. 12A and 12B).In addition to its effect on development, IL-15 also promotes thematuration of NK cells. Consistently, it was found that surfaceexpression of the maturation marker CD16 and the amounts of the lyticgranule protein perforin were higher on NK cells from MISTRG compared toNSG (FIG. 13C to 13F).

The cellular source of IL-15 trans-presentation in vivo in humans iscurrently unknown, but human myeloid cells can support human NK cellproliferation in vitro (Huntington et al., 2009, Journal of experimentalmedicine 206, 25). To test if trans-presentation of human IL-15 by humanmonocytes/macrophages underlies the improved human NK cell developmentin MISTRG, the mice were treated with liposome-encapsulated clodronateto deplete phagocytic cells (FIG. 14). The depletion of phagocytic cellsalso induced a significant reduction of human NK cells (FIG. 3E),suggesting that human monocytes/macrophages are indeed a critical celltype that trans-presents IL-15 to support human NK cell homeostasis invivo.

NK cells participate in the innate defense against pathogens by killingcells that lack the expression of MHC class I (missing-self) (Raulet,2006, Seminars in immunology 18, 145), and by producing the key cytokineIFNγ (Vivier et al., 2008, Nature immunology 9, 503). Consistent withhigher perforin expression (FIGS. 13E and 13F), significantly enhancedNK cell cytotoxic activity against human cells lacking MHC class I wasobserved in vivo in MISTRG compared to NSG (FIG. 3F). NK cells are anearly source of IFNγ after Listeria infection. Accordingly, it was foundthat expression of human IFNγ mRNA in the liver was more than 10-foldhigher in MISTRG than in NSG two days post-infection (FIG. 3G). Atsingle-cell resolution, NK cells from Listeria-infected MISTRG showedproduction of human IFNγ without ex vivo restimulation (FIG. 3H), atfrequencies significantly higher than in NSG (FIG. 3I). NK cells inMISTRG also had lytic activity (degranulation) after Listeria infection,as shown by plasma membrane exposure of CD107a (FIG. 3H). Overall,MISTRG via efficient production of human myeloid cells support thedevelopment, differentiation, and function of human NK cells, therebyovercoming one major limitation of current HHLS mouse models.

Next, the role of human myeloid cells in the context of a tumormicroenvironment was assessed. Therefore, the human melanoma cell lineMe290 was used as a tumor model (Valmori et al., 1998, Journal ofimmunology 160, 1750). Clinical observations show that myeloid cellsinfiltrate tumors in several solid tumors, and high densities ofinfiltrating macrophages correlate with poor patient prognosis in mosttypes of cancer (Qian and Pollard, 2010, Cell 141, 39; Coussens et al.,Science 339, 286; Egeblad et al., 2010, Developmental cell 18, 884;Nelson and Bissell, 2006, Annual review of cell and developmentalbiology 22, 287; Bingle et al., 2002, T Journal of pathology 196, 254).Accordingly, higher human myeloid cell infiltration was detected intumors in MISTRG than in NSG, as shown by the expression of human PTPRCand ITGAM mRNA (encoding respectively CD45 and CD11b) (FIG. 4A). Closelyresembling human tumors in patients, cells expressing the macrophagemarkers CD163 and CD14 were abundant in tumors in MISTRG, but werealmost undetectable in the same tumors in NSG (FIGS. 4B and 4C; and FIG.15). Most of the CD163+ cells also expressed low levels of HLA-DR andhigh levels of CD206 (FIGS. 4B and 4D), an immunophenotype generallyassociated with “M2-like” macrophages (Hao et al., 2012, Clinical &developmental immunology 2012, 948098; Tang, 2013, Cancer Lett 332, 3).

The M2 subtype of macrophages promotes tumor progression via a varietyof effector mechanisms, including proliferative signals to cancer cells,anti-apoptotic signals, pro-angiogenic activity, enabling cancer cellegress from primary tumors and formation of metastasis (Qian andPollard, 2010, Cell 141, 39; Coussens et al., Science 339, 286; Egebladet al., 2010, Developmental cell 18, 884). Macrophage infiltration intumors could promote tumor growth in MISTRG was assessed. Remarkably, itwas observed that the size of the tumors in CD34+-engrafted MISTRG,which are heavily infiltrated by human CD163+HLA-DRlow CD206+macrophages, was significantly greater than tumors in NSG, which are notinfiltrated by human macrophages and are the same small size seen innon-engrafted NSG or MISTRG mice (FIGS. 4E and 4F). One of themechanisms by which macrophages support tumor growth is through theproduction of cytokines or enzymes that promote vascularization andimmune suppression. VEGF is an important polyfunctional tumor-supportingmolecule (Kandalaft et al., Current topics in microbiology andimmunology 344, 129; Motz and Coukos, Immunity 39, 61), and to testwhether this factor was involved in tumor growth in MISTRG, the micewere treated with the human-VEGF inhibitor Avastin™. This treatmentcompletely reversed the tumor-growth phenotype (FIG. 4F), demonstratingthat myeloid cells in MISTRG support melanoma growth through aVEGF-dependent mechanism. Overall, these results show that MISTRG micerecapitulate the role of human macrophages in tumor development andfulfill a critical need for models allowing studies of the interactionbetween human tumors and human macrophages in vivo, especially at onsetof tumor development.

The data described here have demonstrated that the provision of multiplehuman cytokines in MISTRG mice resulted in synergistic effects (FIG. 16)on human hematopoiesis and on direct or indirect support for humanimmune cell function. The MISTRG model of HHLS mice offers a uniqueopportunity to study human innate immune responses in vivo.

The materials and methods are now described.

Mouse Strains

The generation of mice with knockin replacement of the genes encodingTPO, IL-3/GM-CSF and M-CSF or with BAC-transgenic expression of humanSIRPα in the RAG2−/−γc−/− Balb/c×129 genetic background was reported(Rathinam et al., 2011, Blood 118, 3119; Willinger et al., 2011,Proceedings of the National Academy of Sciences 108, 2390; Rongvaux etal., 2011, Proceedings of the National Academy of Sciences 108, 2378;Strowig et al., 2011, Proceedings of the National Academy of Sciences108, 13218). These strains were crossbred to obtain MITRG(M-CSFh/hIL-3/GM-CSFh/hTPOh/hRAG2−/−γc−/−) and MISTRG(M-CSFh/hIL-3/GM-CSFh/hhSIRPAtgTPOh/hRAG2−/−γc−/−) mice. Those mice areviable, healthy and fertile. The mice were maintained under specificpathogen free conditions with continuous treatment with enrofloxacin inthe drinking water (Baytril, 0.27 mg/ml). NOD Scid γc−/−(NSG) mice wereobtained from Jackson Laboratory.

Human HSPC Preparation and Engraftment into Recipient Mice

Recipient mice were engrafted with human hematopoietic stem andprogenitor cells as described (Rathinam et al., 2011, Blood 118, 3119;Willinger et al., 2011, Proceedings of the National Academy of Sciences108, 2390; Rongvaux et al., 2011, Proceedings of the National Academy ofSciences 108, 2378; Traggiai et al., 2004, Science 304, 104; Strowig etal., 2011, Proceedings of the National Academy of Sciences 108, 13218).Fetal liver samples were cut in small fragments, treated for 45 min at37° C. with Collagenase D (Roche, 100 ng/mL) and a cell suspension wasprepared. Human CD34+ cells were purified by density gradientcentrifugation (Lymphocyte Separation Medium, MP Biomedicals) followedby positive immunomagnetic selection with anti-human CD34 microbeads(Miltenyi Biotec). Cells were frozen in FBS containing 10% DMSO and keptin liquid nitrogen.

For engraftment, newborn pups (within first 2 days of life) weresublethally irradiated (X-ray irradiation; RG, 2×180 cGy 4 h apart; NSG,1×100 cGy; MISTRG, 1×150 cGy) and 100,000 FL-CD34+ cells in 20 μL of PBSwere injected into the liver with a 22-gauge needle (Hamilton Company).In specific experiments (FIGS. 1D and 1E), 200,000-300,000 cells wereinjected into non-irradiated MISTRG newborn recipients. The mice werebled 7-9 weeks later and the percentage of human CD45+ cells wasmeasured by flow cytometry. Mice in which human CD45+ cells representedat least 5% (RG) or 10% (NSG, MITRG and MISTRG) of the total (mouse andhuman combined) CD45+ populations were selected for furtherexperimentation. The mice were sacrificed or used for experiments 9-12weeks after transplantation.

All experiments were performed in compliance with Yale University HumanInvestigation Committee and Yale Institutional Animal Care and UseCommittee protocols.

Immunophenotypic Analysis of Human Cell Populations

To prepare WBCs, heparinized blood was treated twice with ACK lysisbuffer to eliminate RBCs. Single cell suspension of the spleen and bonemarrow (flushed from the femur and tibia) were treated with ACK lysisbuffer. Liver and lung leukocytes were isolated by mechanicallydissociating and digesting tissues with 100 U/ml collagenase IV and 0.02mg/ml DNase I (Sigma) for 1 h at 37° C., followed by density gradientcentrifugation.

For FACS analysis, antibodies against the following antigens were used:

Mouse antigens: CD45 (clone 30-F11), CD71 (RI7217), Ter119

Human antigens: CD1c (BDCA1, clone L161), CD3 (UCHT1), CD11b (ICRF44),CD11c (3.9), CD14 (M5E2), CD16 (3G8), CD19 (HIB19), CD33 (WM53), CD45(HI30), CD62L (DREG-56), CD66 (ASL-32), CD94 (DX22), CD107a (H4A3),CD115 (9-4D2-1E4), CD123 (6H6), CD141 (BDCA3, M80), CD161 (HP-3G10),CD235a (HI264), CD303 (BDCA2, 201A), NKp46 (9E2), IL-15Rα (JM7A4),CX3CR1 (2A9-1), HLA-A,B,C (W6/32), HLA-DR (L243), IFNγ (B27) KIR2DU/S1(HP-MA4), KIR2DL2/L3 (DX27), KIR3DL1 (DX9), perforin (dG9).

Human lineage cocktail: CD3, CD15, CD19, CD56, NKp46

All antibodies were obtained from Biolegend, BD Biosciences or MiltenyiBiotec. Data were acquired with FACSDiva on a LSRII flow cytometer (BDBiosciences) and analyzed with FlowJo software.

For histological analysis, spleen, lung, liver and colon tissues werefixed overnight in IHC zinc fixative (BD Biosciences) or 4%paraformaldehyde and embedded in paraffin. Sections were stained withhematoxylin and eosin, or with anti-human CD68 antibody (clone PGM1)followed by a HRP-conjugated secondary antibody and revealed with theperoxidase substrate 3, 3′-diaminobenzidine.

Phagocytosis Assay In Vitro

E. Coli expressing GFP were grown in LB medium overnight at 37° C. to anOD600 of 1.5-1.8, at which point the bacteria were diluted and grown for1-2 hours to an OD600 of approximately 1.0. The E. Coli were washedthree times with PBS and incubated with WBCs from MITRG mice for 4 hoursat 37° C. in a volume of 200 μl with about 2×10⁸ E. Coli per 1×10⁷ WBCs.After the incubation, the cells were washed with PBS and analyzed byflow cytometry.

TLR Stimulation In Vitro and Infection In Vivo

Human monocyte subsets were isolated from the BM of mice. Briefly, BMcells were recovered and pooled from the hind legs and the spine of sixmice. Human CD33+ cells were enriched by magnetic isolation (EasySepCD33 selection kit, StemCell Technologies). CD14+CD16- and CD14+CD16+subsets were purified on a FACSAria cell sorter (BD Biosciences).100,000 cells in 200 μl media were cultivated overnight in the presenceof the TLR4 ligand LPS (E. Coli 0111:B4, Sigma-Aldrich, 100 ng/ml) orthe TLR7/8 ligand R848 (Invivogen, 10 μg/ml).

For in vivo stimulation, 35 μg of LPS (E. Coli 0111:B4, Sigma-Aldrich)in 100 μl PBS were injected intra-peritoneally and the serum wascollected 90 minutes later.

Mice were infected with 3×10³ colony-forming units (CFU) of Listeriamonocytogenes (strain 10403S) by intravenous injection. Forty-eighthours after infection, sera and tissues were harvested for ELISA andqPCR, respectively. Liver lymphocytes from uninfected or infected micewere incubated at 37 C°/5% CO₂ for 4 hours in medium containing monensin(Golgi Stop, BD Biosciences) and anti-human CD107a antibody. Cells werethen stained for surface antigens, permeabilized using Cytofix/Cytopermkit (BD Biosciences), and stained for intracellular human IFNγ.

Mice were infected intranasally with 2×10⁴ PFU of influenza A/PR8 (H1N1)virus, and lungs were harvested on day 3 postinfection for qPCRanalysis.

Cytokine concentrations (human TNFα, IL-6 and IL-1β) in mouse serum andin culture supernatants were measured using ELISA MAX Standard kits(Biolegend), following the manufacturer's instructions.

RBC Analysis

RBC counts were measured on a Hemavet 950 (Drew Scientific). Bloodsmears were stained with Wright-Giemsa. For mouse RBC transferexperiments, blood was obtained from RG mice, labeled with CFSE (20 μM,15 minutes at 37° C.), washed three times with PBS and 200 μl of labeledRBCs were injected by retro-orbital intravenous injection. The mice werebled 5 minutes later to determine the initial frequency (Day 0, 100%) ofCFSE-positive cells among Ter119+ cells by flow cytometry. They werethen bled at the indicated time points and the maintenance ofCFSE-labeled Ter119+ cells was calculated as a percentage of Day 0values.

Depletion of Phagocytic Cells In Vivo

Phagocytic cells were depleted by intravenous retro-orbital injection of100 μl of clodronate-loaded liposomes (Van Rooij en and Sanders, 1994,Journal of immunological methods 174, 83). Clodronate-liposomes wereinjected 3 times daily and human NK cells in mouse liver were analyzed24 h after the last injection. For RBC phagocytosis assay,clodronate-liposomes were injected 3 days and 1 day prior to transfer ofCFSE-labeled RBCs.

Quantitative RT-PCR

Total RNA was extracted from tissues or purified cells with TRIzolreagent (Invitrogen) according to the manufacturer's instructions andused for cDNA synthesis with the SuperScript First-Strand SynthesisSystem (Invitrogen). Quantitative RT-PCR was performed on a 7500 FastReal-Time PCR system with primer-probe sets purchased from ABI.Expression values were calculated using the comparative threshold cyclemethod and normalized to mouse Hprt or human HPRT, as indicated.

In Vivo NK Cell Cytotoxicity Assays

Human NK cell cytotoxicity in vivo was determined following a previouslyreported protocol (Strowig et al., 2010, Blood 116, 4158). LCL721.221(HLA class I negative) and LCL721.45 (class I positive) cells were mixedin a 1:1 ratio, labeled with CellTrace Violet (Invitrogen) and injectedintravenously (1×10⁷ cells/mouse) into engrafted NSG or MISTRG mice.Mice were sacrificed 12 hours later and single cell suspension of thespleens were prepared and analyzed by flow cytometry. The proportions ofHLA class I positive and negative among violet cells were measured andspecific lysis was calculated as (MHC class I positive—MHC class Inegative)×100/MHC class I positive.

Tumorigenesis

The human melanoma cell line Me290 (Valmori et al., 1998, Journal ofimmunology 160, 1750) was grown to ˜90% confluency and the cells (˜7million cells per mouse) were injected subcutaneously under anesthesiain the flank of the mouse. For some experiments, the mice were treatedevery other day, starting on the day of tumor implantation, with theanti-human VEGF antibody Avastin™ (Roche; 100 μg intravenously). Thesize of the tumors was measured 11 days later and the volume calculatedusing the following formula: Volume=0.5*Length2*Width.

Patients and mouse tissues were frozen in Optimum Cutting Temperature(OCT, Sakura Finetek). Cryosections (7 μm) were consecutively treatedwith Triton-100× 0.1% for 15 min, Hyaluronidase 0.03% for 15 min,Background Buster (Innovex bioscience) for 15 min, Fc Receptor Block(Innovex bioscience) for 15 min and Background Buster for an additional15 min. The sections were then stained with primary antibodies, dilutedin PBS supplemented with 5% BSA and 0.01% Saponin for 1 h at roomtemperature, washed and stained with the secondary antibodies at roomtemperature for 40 minutes. Nuclei were stained with4′,6-diamidino-2-phenylindole (1 μg/mL) for 2 min.

Primary antibodies: human CD14 (1:200, UCHM1, AbD Serotec); human CD163(1:200, EDHu-1, AbD Serotec); human CD206 (1:100, 15-2, AbD Serotec);human HLA-DR (1:100, LN3, Biolegend). For CD163/CD206 combined staining,both antibodies were labeled with Alexa Fluor 488 or 568 AntibodyLabeling Kit (Molecular Probes) prior tissue staining.

Secondary antibodies: goat anti-rat Alexa Fluor 568; goat anti-mouseAlexa Fluor 488; goat anti-mouse Alexa Fluor 588 or goat anti-mouseAlexa Fluor 647 (1:700, Molecular Probes).

Immunofluorescence imaging was performed on an Eclipse Ti invertedmicroscope system (Nikon Instruments Inc.) operated via NIS-Element Arsoftware (Nikon Instruments Inc).

For quantification of the density of CD163+ cell infiltration, tumorsfrom 3 different melanoma patients, 3 NSG and 3 MISTRG were selected.From each tumor, 3 cryosections were stained for human CD163. From eachstained section 3 representative pictures were acquired, totaling 27representative pictures from each group (Patients, MISTRG and NSG). Foreach picture, CD163+ cells were counted using the NIS-Element Arsoftware (Niko Instruments Inc.). Each picture was analyzed using the“split channels+overlay” display and by zooming simultaneously on eachseparate channel and on the overlay.

Statistical Analysis

Statistical analysis was performed with the GraphPad Prism 5 software,using one-way ANOVA followed by Tukey post hoc test, two-tailed unpairedStudent's t-test or repeated measure ANOVA.

Example 2: Human Myeloid Neoplasms can be Engrafted in MISTRG

Myeloid leukemia is a form of cancer that affects cells of the myeloidlineage. Myeloid leukemias are classified in different types, includingacute myeloid leukemia (AML), myeloproliferative disorder (MPD), chronicmyelo-monocytic leukemia (CMML) and myelodysplastic syndrome (MDS). Therisk of developing myeloid leukemias increases with age and theincidence of these diseases is likely to increase with ageing of thepopulation. Although therapeutic and supportive care approaches areavailable in the clinic, a better understanding of this group ofdiseases and novel therapies are needed.

One of the methods used to study human leukemias relies on thexeno-transplantation of patient samples into immunodeficient mice.However, currently available recipient mice are not optimal for thispurpose: only a subset of AML samples can be engrafted successfully; androbust engraftment of MPD, CMML or MDS (including RCUD, RAEB I and RAEBII) has not been reported so far. Thus, optimized strains of recipientmice are needed for better engraftment of human myeloid leukemia.

It is demonstrated herein that MISTRG supports better engraftment ofhuman hematopoietic cells, leading to the almost complete replacement ofmouse hematopoiesis by human hematopoiesis in the bone marrow. It isalso shown herein that samples isolated from patients with AML, CMML andMDS can be engrafted in MISTRG (FIGS. 17A-17D).

Therefore, the genetically modified non-human animals described hereinrepresent a novel in vivo animal model of human myeloid leukemia thatwill be useful to (i) study the cellular and molecular pathogenesis ofthe disease; (ii) to identify biomarkers with predictive or prognosticvalue; (iii) to identify novel targets for therapies; and (iv) to testtherapies in a pre-clinical and patient-specific setting.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A genetically modified mouse comprising in its genome: arecombination activating gene 2 (Rag-2) gene knock-out; an IL2 receptorgamma chain (IL2rg) gene knock-out; a replacement of a mouse M-CSF genewith a nucleic acid encoding a human M-CSF polypeptide, wherein thenucleic acid encoding the human M-CSF polypeptide is operably linked toa M-CSF promoter at a mouse M-CSF gene locus; a replacement of a mouseIL-3 gene with a nucleic acid encoding a human IL-3 polypeptide, whereinthe nucleic acid encoding the human IL-3 polypeptide is operably linkedto a IL-3 promoter at a mouse IL-3 gene locus; a replacement of a mouseGM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide,wherein the nucleic acid encoding the human GM-CSF polypeptide isoperably linked to a GM-CSF promoter at a mouse GM-CSF gene locus; areplacement of a mouse-SIRPA gene with a nucleic acid encoding a fusionprotein comprising a biologically active fragment of a full length humanSIRPA polypeptide, wherein the nucleic acid encoding the fusion proteinis operably linked to a SIRPA promoter; and a replacement of a mouse TPOgene with a nucleic acid encoding a human TPO polypeptide, wherein thenucleic acid encoding the human TPO polypeptide is operably linked to aTPO promoter at a mouse TPO gene locus, wherein the mouse expresses thehuman M-CSF polypeptide, the human IL-3 polypeptide, the human GM-CSFpolypeptide, the fusion protein, and the human TPO polypeptide. 2.-8.(canceled)
 9. The genetically modified mouse of claim 1, furthercomprising human hematopoietic cells.
 10. The genetically modified mouseof claim 1, further comprising a human cancer cell.
 11. The geneticallymodified mouse of claim 10, wherein the human cancer cell is a leukemiacell or a melanoma cell.
 12. A method of hematopoietic stem andprogenitor cell (HSPC) engraftment in a genetically modified mouse, themethod comprising the step of: administering at least one HSPC to thegenetically modified mouse, wherein the genetically modified mousecomprises in its genome: a recombination activating gene 2 (Rag-2) geneknock-out; an IL2 receptor gamma chain (IL2rg) gene knock-out; areplacement of a mouse M-CSF gene with a nucleic acid encoding a humanM-CSF polypeptide, wherein the nucleic acid encoding the human M-CSFpolypeptide is operably linked to a M-CSF promoter at a mouse M-CSF genelocus; a replacement of a mouse IL-3 gene with a nucleic acid encoding ahuman IL-3 polypeptide, wherein the nucleic acid encoding the human IL-3polypeptide is operably linked to a IL-3 promoter at a mouse IL-3 genelocus; a replacement of a mouse GM-CSF gene with a nucleic acid encodinga human GM-CSF polypeptide, wherein the nucleic acid encoding the humanGM-CSF polypeptide is operably linked to a GM-CSF promoter at a mouseGM-CSF gene locus; a replacement of a mouse-SIRPA gene with a nucleicacid encoding a fusion protein comprising a biologically active fragmentof a full length human SIRPA polypeptide, wherein the nucleic acidencoding the fusion protein is operably linked to a SIRPA promoter; anda replacement of a mouse TPO gene with a nucleic acid encoding a humanTPO polypeptide, wherein the nucleic acid encoding the human TPOpolypeptide is operably linked to a TPO promoter at a mouse TPO genelocus, wherein the mouse expresses the human M-CSF polypeptide, thehuman IL-3 polypeptide, the human GM-CSF polypeptide, the fusionprotein, and the human TPO polypeptide. 13.-18. (canceled)
 19. Themethod of claim 12, wherein the genetically modified mouse comprises ahuman cancer cell.
 20. The method of claim 19, wherein the human cancercell is a leukemia cell or a melanoma cell. 21.-27. (canceled)
 28. Themethod of claim 12, wherein the administering step is not preceded bypreconditioning irradiation of the genetically modified mouse oradministration of a radiomimetic drug to the genetically modified mouse.29. The method of claim 12, further comprising administering a humancancer cell to the genetically modified mouse.
 30. The method of claim29, wherein the human cancer cell is a leukemia cell or a melanoma cell.31. The method of claim 29, wherein the at least one HSPC and the humancancer cell are both isolated from the same patient.